A dynamic partitioning mechanism polarizes membrane protein distribution

The plasma membrane is widely regarded as the hub of the numerous signal transduction activities. Yet, the fundamental biophysical mechanisms that spatiotemporally compartmentalize different classes of membrane proteins remain unclear. Using multimodal live-cell imaging, here we first show that several lipid-anchored membrane proteins are consistently depleted from the membrane regions where the Ras/PI3K/Akt/F-actin network is activated. The dynamic polarization of these proteins does not depend upon the F-actin-based cytoskeletal structures, recurring shuttling between membrane and cytosol, or directed vesicular trafficking. Photoconversion microscopy and single-molecule measurements demonstrate that these lipid-anchored molecules have substantially dissimilar diffusion profiles in different regions of the membrane which enable their selective segregation. When these diffusion coefficients are incorporated into an excitable network-based stochastic reaction-diffusion model, simulations reveal that the altered affinity mediated selective partitioning is sufficient to drive familiar propagating wave patterns. Furthermore, normally uniform integral and lipid-anchored membrane proteins partition successfully when membrane domain-specific peptides are optogenetically recruited to them. We propose “dynamic partitioning” as a new mechanism that can account for large-scale compartmentalization of a wide array of lipid-anchored and integral membrane proteins during various physiological processes where membrane polarizes.

File Name: Supplementary Movie 2 Description: Consistent dynamic localization of Gβγ into the back-state regions of the membrane.Ventral wave propagation in the substrate-attached surface of Dictyostelium cells co-expressing KikGR-Gβγ and PHCrac-mCherry, demonstrating that complementary distribution between front-state marker PHCrac and Gβγ is highly consistent during dynamic pattern formations.Left Panels: KikGR-Gβγ, middle panels: PHCrac-mCherry, right panels: merged view.Top right-corner: time in mm:ss format.
File Name: Supplementary Movie 3 Description: Consistent dynamic localization of RasG into the back-state regions of the membrane.Ventral wave propagation in the substrate-attached surface of Dictyostelium cells co-expressing GFP-RasG and PHCrac-mCherry, demonstrating that complementary distribution between front-state marker PHCrac and RasG is consistent during dynamic pattern formations.Left Panels: GFP-RasG, middle panels: PHCrac-mCherry, right panels: merged view.Top rightcorner: time in mm:ss format.File Name: Supplementary Movie 6 Description: Uniform distribution of cAR1 on the membrane during ventral wave propagation.Ventral wave propagation in the substrate-attached surface of Dictyostelium cells co-expressing cAR1-GFP and PHCrac-mCherry, demonstrating that cAR1 does not exhibit symmetry breaking and is uniformly distributed on the membrane.Left Panels: cAR1-GFP, middle panels: PHCrac-mCherry, right panels: merged view.Top right-corner: time in mm:ss format.
File Name: Supplementary Movie 7 Description: Dynamic localization of R(+8)-Pre into the back-state regions of the membrane in RAW 264.7 macrophages.Ventral wave propagation in the substrate-attached surface of RAW 264.7 macrophages co-expressing GFP-R(+8)-Pre and PHAkt-mCherry, demonstrating that complementary distribution between front-state marker PHAkt and R(+8)-Pre is highly consistent during dynamic pattern formations which were induced by frustrated phagocytosis and osmotic shock.Left panels: GFP-R(+8)-Pre, middle panels: PHAkt-mCherry, right panels: merged view.Top right-corner: time in mm:ss format.
File Name: Supplementary Movie 8 Description: Dynamic localization of R(+8)-Pre into the back-state regions of the membrane in cytoskeletal dynamics impaired RAW 264.7 macrophages.Ventral wave propagation in the substrate-attached surface of RAW 264.7 macrophages co-expressing GFP-R(+8)-Pre and PHAkt-mCherry, demonstrating that complementary distribution between front-state marker PHAkt and R(+8)-Pre is highly consistent during dynamic pattern formations (i.e., in both larger-scale waves and smaller-scale wavelets), even when actin polymerization was inhibited with 5µM Latrunculin A and ROCK activity was inhibited with 50µM of Y-27632 (se Methods for details).Left panels: GFP-R(+8)-Pre, middle panels: PHAkt-mCherry, right panels: merged view.Top rightcorner: time in mm:ss format.
File Name: Supplementary Movie 9 Description: Kinetics of CynA and PHCrac during global receptor activation.Global cAMP stimulation driven receptor activation in Dictyostelium cells co-expressing CynA-KikGR and PHCrac-mCherry, demonstrating that upon receptor activation front protein PHCrac gets recruited to membrane from cytosol whereas back-associated peripheral membrane protein CynA gets dissociated from membrane and moves to cytosol.Left Panels: CynA-KikGR, right panels: PHCrac-mCherry.Top right-corner: time in seconds.cAMP was added at time t=0s (also indicated by the appearance of white text "+cAMP stimulation" in the video).
File Name: Supplementary Movie 10 Description: Kinetics of PKBR1 and PHCrac during global receptor activation.Global cAMP stimulation driven receptor activation in Dictyostelium cells co-expressing PKBR1-KikGR and PHCrac-mCherry, demonstrating that upon receptor activation front protein PHCrac gets recruited to membrane from cytosol whereas back-associated lipid-anchored protein PKBR1 maintained membrane association.Left Panels: PKBR1-KikGR, right panels: PHCrac-mCherry.Top left-corner: time in seconds.cAMP was added at time t=0s (also indicated by the appearance of white text "+cAMP stimulation" in the video).
File Name: Supplementary Movie 11 Description: Kinetics of Gβγ and PHCrac during global receptor activation.Global cAMP stimulation driven receptor activation in Dictyostelium cells co-expressing KikGR-Gβγ and PHCrac-mCherry, demonstrating that upon receptor activation front protein PHCrac gets recruited to membrane from cytosol whereas back-associated lipid-anchored protein Gβγ maintained membrane association.Left Panels: KikGR-Gβγ, right panels: PHCrac-mCherry.Top left-corner: time in seconds.cAMP was added at time t=0s (also indicated by the appearance of white text "+cAMP stimulation" in the video).
File Name: Supplementary Movie 12 Description: Kinetics of R(+8)-Pre and PHCrac during global receptor activation.Global cAMP stimulation driven receptor activation in Dictyostelium cells co-expressing GFP-R(+8)-Pre and PHCrac-mCherry, demonstrating that upon receptor activation front protein PHCrac gets recruited to membrane from cytosol whereas back-associated lipid-anchored synthetic protein R(+8)-Pre maintained membrane association.Left Panels: GFP-R(+8)-Pre, right panels: PHCrac-mCherry.Top left-corner: time in seconds.cAMP was added at time t=0s (also indicated by the appearance of white text "+cAMP stimulation" in the video).
File Name: Supplementary Movie 13 Description: Kinetics of cAR1 and PHCrac during global receptor activation.Global cAMP stimulation driven receptor activation in Dictyostelium cells co-expressing cAR1-GFP and PHCrac-mCherry, demonstrating that upon receptor activation front protein PHCrac gets recruited to membrane from cytosol whereas uniformly distributed transmembrane protein cAR1 maintained membrane association.Left Panels: cAR1-GFP, right panels: PHCrac-mCherry.Top left-corner: time in seconds.cAMP was added at time t=0s (also indicated by the appearance of white text "+cAMP stimulation" in the video).Scale bar: 10 µm.File Name: Supplementary Movie 15 Description: Selective photoconversion of CynA suggests a shuttling mechanism for its polarized distribution.In the ventral surface of a Dictyostelium cell expressing CynA-KikGR, a membrane domain which is switching from back/basal to front/activated state (i.e. an area right ahead of a "shadow" wave) was photoconverted selectively using a ROI where 405 nm laser was illuminated.Note that photoconverted CynA vanished from the plane of membrane since it translocated to the cytosol, as shadow wave crossed the photoconverted area.Left Panels: CynA-KikGR (green), right panels: photoconverted CynA-KikGR (red, shown in magenta).Top left-corner: time in mm:ss format.Selective photoconversion was started at time t=0s.

File Name: Supplementary Movie 16
Description: Selective photoconversion of PTEN suggests a shuttling mechanism for its polarized distribution.In the ventral surface of a Dictyostelium cell expressing PTEN-KikGR, a membrane domain which is switching from back/basal to front/activated state (i.e., an area right ahead of a "shadow" wave) was photoconverted selectively using a ROI where 405 nm laser was illuminated.Note that photoconverted PTEN, vanished from the plane of membrane since it translocated to the cytosol, as shadow wave crossed the photoconverted area.Left Panels: PKBR1-KikGR (green), right panels: photoconverted PTEN-KikGR (red, shown in magenta).Top left-corner: time in mm:ss format.Selective photoconversion was started at time t=0s.
File Name: Supplementary Movie 17 Description: Selective photoconversion of PKBR1 suggests a partitioning mechanism for its polarized distribution.In the ventral surface of a Dictyostelium cell expressing PKBR1-KikGR, a membrane domain which is switching from back/basal to front/activated state (i.e. an area right ahead of a "shadow" wave) was photoconverted selectively using a ROI where 405 nm laser was illuminated.Note that photoconverted PKBR1, instead of disappearing from membrane and moving to cytosol, rearranged over the plane of membrane.Left Panels: PKBR1-KikGR (green), right panels: photoconverted PKBR1-KikGR (red, shown in magenta).Top leftcorner: time in mm:ss format.Selective photoconversion was started at time t=0s.Two examples are shown.
File Name: Supplementary Movie 18 Description: Selective photoconversion of Gβγ suggests a partitioning mechanism for its polarized distribution In the ventral surface of a Dictyostelium cell expressing KikGR-Gβγ, a membrane domain which is switching from back/basal to front/activated state (i.e. an area right ahead of a "shadow" wave) was photoconverted selectively using a ROI where 405 nm laser was illuminated.Note that photoconverted Gβγ, instead of disappearing from membrane and moving to cytosol, rearranged over the plane of membrane.Left Panels: KikGR-Gβγ (green), right panels: photoconverted KikGR-Gβγ (red, shown in magenta).Top left-corner: time in mm:ss format.Selective photoconversion was started at time t=0s.Two examples are shown.

File Name: Supplementary Movie 19
Description: Single-molecule imaging of PKBR1 and simultaneous PIP3 wave imaging.Singlemolecules of PKBR1-HaloTMR (shown in green) were imaged during ventral wave propagation in a cell, which is also expressing PIP3 sensor PHD-eGFP (shown in magenta).In this multiscale imaging setup, PIP3 sensor was indicating the separate front-state (enriched in PIP3) and backstate (depleted of PIP3) regions, whereas the single-molecules of PKBR1 was recorded to compute its diffusion profiles inside front as well as back state regions.The movies are played back at the same speed as they were taken (30 frames/second).
File Name: Supplementary Movie 20 Description: Two-dimensional Stochastic simulation of an excitable network that incorporated differential diffusion dynamics of lipid-anchored proteins.The spatiotemporal patterns of F, R, B, PP, and LP demonstrate that upon firing of the excitable network, both PP and LP consistently align to asymmetric patterns.In F/B combined panel, F is in green.In all other cases, concentrations are shown in Matplotlib "Plasma" colormap.This video corresponds to Figure 6c.
File Name: Supplementary Movie 21 Description: Two-dimensional Stochastic simulation of an excitable network where differential diffusion dynamics of lipid-anchored proteins was neglected.The spatiotemporal patterns of F, R, B, PP, and LP demonstrates that upon firing of the excitable network, only PP consistently aligns to asymmetric patterns, but LP, due to its uniform diffusion along all grid points, could not undergo symmetry breaking.In F/B combined panel, F is in green.In all other cases, concentrations were shown in Matplotlib "Plasma" colormap.This video corresponds to Supplementary Figure 13a.
File Name: Supplementary Movie 22 Description: Optogenetic recruitment of cytosolic CRY2PHR-mCherry-R+ and cytosolic CRY2PHR-mCherry(CTRL) to membrane bound cAR1-CIBN.First two movies demonstrate two examples of recruitment of cytosolic CRY2PHR-mCherry-R+ to membrane bound cAR1-CIBN demonstrating synthetically increasing affinity for the back-state region is sufficient to generate polarized pattern out of a normally uniformly distributed protein cAR1.Third movie demonstrate that CRY2PHR-mCherry(CTRL) recruitment to cAR1-CIBN does not induce any symmetry breaking.In first two movies: Left panels: CRY2PHR-mCherry-R+.In third movie: Left panels: CRY2PHR-mCherry(CTRL).In all movies: Middle panels: Lifeact-HaloTag (Janelia Flour 646); Right panels: Merged view.Top left corners showing time in second.The 488 nm laser was globally turned on at time t=0s to initiate optogenetic recruitment (also indicated by the appearance of white text "488 nm ON" in the video).

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Supplementary Movie 4 Description: Consistent dynamic localization of synthetic protein PKBR1N150 into the back-state regions of the membrane.Ventral wave propagation in the substrate-attached surface of Dictyostelium cells co-expressing PKBR1N150-KikGR and PHCrac-mCherry, demonstrating that complementary distribution between front-state marker PHCrac and PKBR1N150 is highly consistent during dynamic pattern formations.Left Panels: PKBR1N150-KikGR, middle panels: PHCrac-mCherry, right panels: merged view.Top right-corner: time in mm:ss format.File Name: Supplementary Movie 5 Description: Consistent localization of PKBR1 into the back of the membrane in chemotaxing Dictyostelium cells.Developed Dictyostelium cells, co-expressing PKBR1-KikGR and LimE-mCherry, chemotaxing towards a micropipette filled with 10 µM of cAMP.Left Panels: PKBR1-KikGR, middle panels: LimE-mCherry, right panels: DIC images (which also shows the position of micropipette).Fluorescent channels are shown in matplotlib "Plasma" colormap.Top rightcorner: time in mm:ss format.Scale bar: 10 µm.

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Supplementary Movie 14 Description: Kinetics of R(+8)-Pre and PHAkt during global C5a receptor activation.Two examples of global FKP-(D-Cha)-Cha-r stimulation driven C5a receptor activation in RAW 264.7 cells co-expressing GFP-R(+8)-Pre and PHAkt-mCherry, demonstrating that upon receptor activation front protein PHAkt gets recruited to membrane from cytosol (and eventually the response adapts), whereas back-associated lipid-anchored protein R(+8)-Pre maintained membrane association throughout the experiment.Left Panels: GFP-R(+8)-Pre, right panels: PHAkt-mCherry.Top left-corner: time in seconds.FKP-(D-Cha)-Cha-r was added at time t=0s (also indicated by the appearance of white text "+C5aR agonist" in the video).Scale bars: 10 µm.